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BCR::ABL1-like pre-B-ALL comprises a myriad of genetic lesions making molecular diagnosis challenging and expensive. Its frequency and outcome are less studied in resource-constraint settings.154 pre-B-ALL cases (0-12 years) were enrolled as group 1 (37 cases of B-other-ALL) and group 2 (117 patients with recurrent translocations/ hyperdiploidy). Group 1 was evaluated for BCR::ABL1-like genetic lesions and copy-number abnormalities (CNAs) as per our published PACE approach supplemented with targeted RNA sequencing.BCR::ABL1-like frequency was 5.2% (8 of 154) and 22% (8 of 37) with the PACE approach alone in the whole and B-other-ALL cohort, respectively. The addition of targeted RNA-sequencing had led to the frequency increasing to 9% (14 of 154) and 38% (14 of 37) in the whole and B-other-ALL cohort, respectively. P2RY8::CRLF2, IGH::CRLF2, and RCSD1::ABL1 were noted in 8 (57.1%), 4 (28.6%), and 2 (14.3%) patients, respectively. CNAs were noted in 56.7% (21 of 37) of patients. The BCR::ABL1-like group had a significantly higher initial WBC count of ≥ 50,000/mmThe sensitivity of detecting BCR::ABL1-like lesions had increased significantly from 22% using the PACE approach alone to 38% in B-other-ALLs with the integrated approach. Although outcomes were not statistically different, a higher percentage of relapses were noted in the B-other-ALL group.

Data on childhood acute promyelocytic leukemia (APL) from low-and middle-income countries is limited. Early mortality is a concern and often not highlighted in clinical trials. The retrospective study was conducted on patients (≤12 years) with APL from 2003 to 2021 at a single center in India. Patients were treated with all

NPM1 and FLT3-ITD are frequently mutated genes in acute myeloid leukemia. We studied clinico-hematological profile and survival outcome of adult acute promyelocytic leukemia (APL) patients harboring these mutations.De novo APL cases (12 years), enrolled between January 2019 and June 2020, were evaluated for FLT3-ITD and NPM1 mutations (A, B, D mutations) by conventional and real-time qualitative PCR respectively.FLT3-ITD mutation was detected in 12 of 36 (33.3%) de novo APLs cases while NPM1 mutation was not detected. FLT3-ITD was more frequently associated with Sanz high-risk category as compared to the intermediate-risk category (75% vs. 29%, P = .02), with BCR3 transcript type (P = .08) and higher median WBC count [22.7 × 10FLT3-ITD mutation did not influence survival outcome in adult APL treated with ATO and ATRA-based therapeutic regimen.

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene.The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases.It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

BACKGROUND Early prediction of response to percutaneous catheter drainage (PCD) of necrotic collections in acute pancreatitis (AP) using simple and objective tests is critical as it may determine patient prognosis. The role of white blood cell (WBC) count and neutrophil-lymphocyte ratio (NLR) has not been assessed as a tool of early prediction of PCD success and is the focus of this study. AIM To assess the value of WBC and NLR in predicting response to PCD in AP. METHODS This retrospective study comprised consecutive patients with AP who underwent PCD between June 2018 and December 2019. Severity and fluid collections were classified according to the revised Atlanta classification and organ failure was defined according to the modified Marshall Score. WBC and NLR were monitored 24 h prior PCD (WBC-0/NLR-0) and 24 h (WBC-1/NLR-1), 48 h (WBC-2/NLR-2) and 72 h (WBC-3/NLR-3) after PCD. NLR was calculated by dividing the number of neutrophils by the number of lymphocytes. The association of success of PCD (defined as survival without the need for surgery) with WBC and NLR was assessed. The trend of WBC and NLR was also assessed post PCD. RESULTS One hundred fifty-five patients [median age 40 ± 13.6 (SD), 64.5% males, 53.5% severe AP] were included in the final analysis. PCD was done for acute necrotic collection in 99 (63.8%) patients and walled-off necrosis in 56 (36.1%) patients. Median pain to PCD interval was 24 ± 69.89 d. PCD was successful in 109 patients (group 1) and 46 patients (group 2) who failed to respond. There was no significant difference in the baseline characteristics between the two groups except the severity of AP and frequency of organ failure. Both WBC and NLR showed an overall decreasing trend. There was a significant difference between WBC-0 and WBC-1 (P = 0.0001). WBC-1 and NLR-1 were significantly different between the two groups (P = 0.048 and 0.003, respectively). The area under the curve of WBC-1 and NLR-1 for predicting the success of PCD was 0.602 and 0.682, respectively. At a cut-off value of 9.87 for NLR-1, the sensitivity and specificity for predicting the success of PCD were calculated to be 75% and 65.4% respectively. CONCLUSION WBC and NLR can be used as simple tests for predicting response to PCD in patients with acute necrotizing pancreatitis.

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes.

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) form the backbone of the treatment of acute promyelocytic leukaemia (APL), with the addition of chemotherapy for high-risk patients. We describe our experience of treating patients with APL of all risk classes with ATO and ATRA without chemotherapeutic agents. Patients received induction with ATO and ATRA followed by three cycles of consolidation with ATO and ATRA (each 1 month apart) after achieving morphological remission. Patients with intermediate- and high-risk disease received a further 2 years of maintenance with ATRA, 6-mercaptopurine and methotrexate. A total of 206 patients were included in the study. The majority of the patients were intermediate risk (51.9%), followed by high risk (43.2%). Differentiation syndrome was seen in 41 patients (19.9%). Overall, 25 patients (12.1%) died within 7 days of initiating therapy. Seven patients relapsed during follow-up. The mean (SD) estimated 5-year event-free survival (EFS) and overall survival (OS) in the entire cohort was 79% [5.8%] and 80% [5.8%] respectively. After excluding patients who died within 7 days of therapy initiation, the mean (SD) estimated 5-year EFS and OS was 90% [5.8%] and 93% [3.9%] respectively. Our study shows that treatment of all risk classes of APL with ATO and ATRA without chemotherapy is associated with excellent long-term outcomes in the real-world setting.

Pediatric HIV is a significant contributor to childhood morbidity and mortality. As HIV infects bone marrow CD34-positive progenitor cells, the hematological abnormalities are well-expected. The viral load is much higher in children predisposing them to opportunistic infections and hematopoietic malignancies. The present study aimed to determine the spectrum of hematological abnormalities that may yield clinically useful information in HIV-infected children. This was a retrospective study of 19 HIV-infected children who underwent bone marrow examination. Overall, 20 peripheral blood and bone marrow samples were analyzed for morphological changes in all hematopoietic lineages. Immunohistochemistry was performed on trephine biopsy sections for evaluation of B and T cells and cytomegalovirus inclusions. Anemia was the most common cytopenia (95%), followed by thrombocytopenia (45%) and leukopenia (40%). Myelodysplasia was noted in 70% of cases. Dysmegakaryopoiesis was predominant in 65% of cases, while dyserythropoiesis was noted in 25% of cases. Hemophagocytosis and reactive cellular changes were noted in 20% of cases each. Benign lymphoid aggregates and granulomatous inflammation were observed in 15% and 10% of cases, respectively. Significant myelofibrosis was found in 35% of cases. One case showed infiltration by Burkitt lymphoma. Parvoviral inclusions were identified in 1 case. An interstitial increase and aggregates of CD8 + T cells and reduced CD4 + T cells were noted. CD20 + B cells were normal to mildly increased in the bone marrow. Bone marrow examination provides clinically significant information in pediatric HIV revealing the underlying cause of hematopoietic abnormalities and allows the exclusion of major hematological neoplasms.

Objective/background B-cell neoplasms are clonal tumors of B cells at various stages of maturation, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic lymphoma (CLL), Burkitt lymphoma (BL), lymphoplasmacytic lymphoma (LPL)/Waldenstrom’s macroglobulinemia (WM), splenic marginal zone lymphoma (SMZL), nodal marginal zone lymphoma (NMZL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL). In this study, we analyzed the frequency of MYD88 L265P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasms. Methods A total of 110 consecutive cases of B-cell neoplasms showing peripheral blood and/or bone marrow infiltration were included. MYD88 L265P mutation was detected by polymerase chain reaction amplification of exon 5 of MYD88 gene, followed by restriction fragment length polymorphism analysis. Results Among the 110 cases, the major group was of CLL (54.5%, n = 60), followed by HCL. Other cases included MCL, LPL, DLBCL, SMZL, NMZL, FL, and BL. MYD88 L265P mutation was seen in 21 (19.1%) cases of B-cell neoplasm, whereas 89 (80.9%) cases were negative for MYD88 L265P mutation. It was most commonly seen in LPL/WM cases followed by HCL, SMZL, CLL, and MCL cases. No case of DLBCL, FL, and BL showed MYD88 L265P mutation. Statistically significant difference was seen for hemoglobin level in CLL cases, with MYD88 L265P mutated cases showing higher mean hemoglobin levels than MYD88 wild-type cases (p = .001). For other parameters, no statistically significant difference was noted between mutated and unmutated cases. Conclusion MYD88 L265P mutation is seen in various B-cell neoplasms; it is most commonly seen in LPL/WM cases but not specific for it.

Calreticulin (CALR) mutations are included in the WHO diagnostic criteria of Myeloproliferative Neoplasm (MPN). Presence of CALR mutation, aids in their diagnosis by excluding reactive causes. There are several molecular techniques to detect CALR mutations; however, these require advanced equipment and technical expertise. An in-house “polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP)” method, using restriction enzymes MseI and MluCI for detection of CALR type-1 and type-2 mutations, was developed. PCR-RFLP method was validated by comparing its results with real-time PCR (RQ-PCR) using CE-IVD marked Ipsogen® CALR RGQ PCR (Qiagen, Germany) kit. All PCR-RFLP positive CALR type-1 and type-2 mutations samples of MPN (n=26) were confirmed positive by RQ-PCR, and all negative samples (n=21) were confirmed to be negative, showing 100% diagnostic sensitivity and specificity for CALR mutation detection by PCR-RFLP. The developed PCR-RFLP method can be used for detection of CALR type-1 and type-2 mutations with only a PCR machine and gel electrophoresis system. It is an inexpensive and easily applicable alternative method which can be adopted even by a small molecular laboratory for detection of CALR mutations.

B-Acute lymphoblastic leukemia (B-ALL) with intracytoplasmic inclusions in lymphoblasts is an unusual presentation. Here, we present a pediatric case of B-ALL with ETV6-RUNX1 rearrangment and unusual blast morphology showing distinct intracytoplasmic eosinophilic inclusions. The case is reported for its rare occurrence, specially in childhood and its persistence in the post-induction marrow aspirate, which subsequently showed poor initial treatment response despite the presence of good prognostic molecular markers. The current literature is reviewed.

Background Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, which is attributed to differences in the genetic characteristics of the leukemic clone. We studied the genomic profile of 52 treatment-naive CLL patients. Methods Genetic analysis was performed by multiplex ligation-dependent probe amplification (MLPA) using the SALSA P038 Probemix (MRC Holland, Amsterdam), which contains probes for 2p (MYCN,ALK,REL), 6q, 8p (TNFRSF10A/B), 8q (EIF3H,MYC), 9p21 (CDKN2A/B), 10q (PTEN), 11q (ATM, RDX, PPP2R1B, CADM1), chromosome 12, 13q14 (RB1, DLEU1/2/7, KCNRG, MIR15A), 14q, 17p (TP53) and chromosome 19, and for NOTCH1 7541-7542delCT, SF3B1 K700E, and MYD88 L265P mutations. Results The median age was 65 years (male:female=2:1). The median hemoglobin, total leukocyte, and platelet counts were 12.4 g/dL, 57.7×109/L, and 176.5×109/L, respectively. At least one genetic abnormality was observed in 34 (65%) patients. The most common abnormality was del(13q14) (deleted DLEU2 and DLEU1/RB1 genes), which was observed in 22 (42%) cases, followed by trisomy 12 [7 (13%) cases]. Del(11q) (deleted ATM, RDX11/PPP2R1B-4) and del(17p) (deleted TP53) were present in 5 (10%) and 2 (4%) cases, respectively. 19p13.2 (CDKN2D-2) amplification and NOTCH1 mutation were found in one case each. Conclusion Genetic abnormalities are commonly (65%) observed in CLL patients. Del(13q), which is associated with DLEU2 and DLEU1/RB1 gene deletion, was the most common. Compared with other abnormalities, del(11q) and del(17p) patients presented with cytopenia and higher Binet stage, while those with del(13q14) had a longer time to first treatment.

Cytogenetic abnormalities (CAs), one of the strongest influencers of therapeutic outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), can be identified by different techniques. Despite several technological advances, many centers with resource-limited settings continue to use either reverse-transcriptase polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) to identify prognostically relevant CAs. We evaluated a simple and cost-effective triple-probe FISH strategy on air-dried blood and bone-marrow smears and compared its performance with a multiplex RT-PCR-based approach in the prognostication of pediatric BCP-ALL patients. Three hundred twenty BCP-ALL patients were tested prospectively and in parallel by FISH on air-dried blood or bone-marrow smears and RT-PCR. The FISH strategy correctly diagnosed all genetic abnormalities identified by RT-PCR. Prognostically relevant genetic abnormalities were missed by RT-PCR in 24 (8.1%) patients. In another 20 children (6%), with samples inadequate for RT-PCR testing (dry taps or due to poor sample quality), a successful FISH testing could be performed on bone-marrow aspirate or trephine-imprint smears. In addition, FISH detected ploidy changes, which could be confirmed by FxCycle Violet-based flow-cytometry. FISH testing on air-dried smears identified more prognostically relevant CAs, provided information on the ploidy status, and could be successfully performed in children with difficulty in bone-marrow sampling.

Splanchnic vein thrombosis is an uncommon life-threatening form of venous thrombosis. It is one the common complication among MPN’s. In the western studies the prevalence of JAK2V617F mutation among SVT patient is high and ranges from 7 to 59%. The frequency of this mutation among Indian SVT patients is heterogenous. This was a prospective case control study. A total 52 cases of SVT and 40 controls were screened for JAK2V617F mutation along with other routine thrombophilic risk factors. Out of total 52 cases, 10 had BCS, 2 had MVT and rest 40 were of PVT/EHPVO. The JAK2V617F mutation was seen in two cases and not in controls. Among the thrombophilic markers, heterozygous FVL mutation, PC, PS and presence of APA were seen in 2, 3, 1 and 3 cases respectively. In addition, eight cases also showed deranged risk factors (5 inherited and 3 acquired), however the repeat testing was not performed due to loss of follow up. Among controls, one person showed presence of APA and one person showed multiple thrombophilic risk factor deficiency. JAK2V617F mutation was observed in 3.8% among north Indian SVT patients. The frequency of mutation is on the lower side as compared to the available Indian data. The other thrombophilia markers (both inherited and acquired) are more frequent (18%) and patients should be routinely screened for these thrombophilia markers.

With newer techniques, improvements have been made in reticulocyte counting, but variability is still there. In this study, we evaluated the three methods of reticulocyte counting, namely automated, flow cytometry, and manual methods for reticulocyte count and also for the enumeration of immature reticulocyte fraction (IRF). Reticulocyte count was done by manual method (light microscopy); two different automated analyzers, LH-780, Beckman Coulter, USA and Pentra-XLR, Horiba Ltd., Japan; and flow cytometry (FACS Canto II, B.D. Bioscience, USA). Also, the classification of stages of maturation was done, and percentage was counted based on quantity of reticulum and its distribution in the cytoplasm. Statistical analysis was done to compare statistical difference between methods. A total of 302 patient samples and 40 normal samples were included. For all automated methods, there is a tendency to overestimate with respect to microscopic methods. There is a strong correlation between manual and automated methods as well as among the 2 automated analyzers. The median IRF in LH-780 had a tendency to be on the higher side as compared with that in Pentra-XLR and manual method (p

Imatinib-induced tyrosine kinase inhibition extends beyond the BCR-ABL mutation, resulting in adverse effects. We evaluated hypogammaglobulinemia as a potential 'off-target' action of imatinib in children with CML. A cross-sectional, observational study was performed. Patients with CML in chronic phase, age18-years at diagnosis, receiving imatinib for a duration exceeding 6-months were enrolled. Serum immunoglobulin G, A, and M were measured by end-point nephelometry. Thirty patients were enrolled. The mean age at diagnosis was 10.4 ± 3.1 years (range: 5-18). The mean age at enrollment was 16.4 ± 4.1 years (range: 9-23). The median dose of imatinib was 287.5 mg/m

The presence of lymphoid nodule in the bone marrow biopsy needs to be differentiated as to whether it is benign or malignant in nature. Several morphological criteria have been suggested to differentiate benign from neoplastic nodules. However, the distinction may not always be clear. Recently, it has been shown that when morphology is taken together with immunohistochemistry, differentiation of benign from neoplastic nodule can be suggested. Although, in present times, ancillary techniques of flow cytometry and molecular diagnostics are available, the importance of morphologic analysis along with immunohistochemistry cannot be undermined. We report a case of IgM monoclonal gammopathy of undetermined significance with reactive lymphoid nodules, differentiated using morphology and immunohistochemistry.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (SC) neoplasm diagnosed by the demonstration of t(9;22)(BCR-ABL1) fusion gene. We performed a flow cytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for CML and its utility for detection of residual disease in CML patients on therapy.Patients with clinical suspicion of CML/CML on follow-up were included, and peripheral (PB) and/or bone marrow (BM) samples were utilized for flow cytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. A pre-titrated antibody cocktail containing CD45, CD34, CD38, and CD26 MoABs was used.A total of 104 samples (63 PB and 41 BM) from 64 patients [suspected CML (n = 30), CML on follow-up (n = 15), and non-CML (n = 19)] were tested. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (median = 0.07 (range 0.002%-26.79%)). None of the patients in the control group (non-CML) and follow-up patients with negative reverse transcriptase-polymerase chain reaction (RT-PCR) results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r = .917).Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust, and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is irrespective of BCR-ABL1 transcript type, and its role in residual disease monitoring needs thorough investigation.

Introduction Despite the impressive responses achieved with tyrosine kinase inhibitor (TKI) therapy, treatment resistance develops in 16-33% of patients of chronic myelogenous leukemia (CML). Of the BCR-ABL1 dependent mechanisms, mutations in the tyrosine kinase domain (TKD) are the commonest cause of resistance. Material and methods Allele specific oligonucleotide - polymerase chain reaction (ASO-PCR) was done for testing the six common TKD mutations, T315I, G250E, E255K, M244V, M351T, and Y253F. Results and conclusion TKD mutation study was done on 83 patients. Of these 44 (53%) were positive for one or more mutations. On analyzing specific mutations, E255K was the commonest mutation seen in 24 (29%) cases, followed by T315I in 23(28%) cases. Y253F mutation was not seen in the present study sample. In the present cohort of 83 patients, 29 (35%) cases were positive for single mutation, 12 (14%) had two mutations and 3 (4%) had three mutations.

Cannibalism is a type of “cell-in-cell” phenomenon commonly described in myeloid lineage malignancies. Although lymphocytes and their precursors are inherently non-phagocytic, there are sporadic case reports describing cannibalism by leukemic lymphoblasts. In the current manuscript, we report the case of a pediatric B-lineage acute lymphoblastic leukemia (B-ALL) patient showing cannibalistic lymphoblasts and have reviewed the clinical and laboratory characteristics of similar cases documented in literature. Our manuscript highlights that all reported B-ALL patients showing cannibalistic lymphoblasts had intra-cytoplasmic vacuolations, and all such treatment-naïve pediatric patients were ETV6-RUNX1 fusion positive and had aberrant expression of myeloid lineage antigens.